Method of inhibiting cell proliferation using an anti-oncogene protein

ABSTRACT

The present invention provides a novel method of inhibiting the growth of tumor cells based upon the discovery that p19 ARF  acts as a suppressor of oncogenic transformation by binding to the MDM2 oncoprotein and blocking MDM2&#39;s ability to target associated proteins, such as p53 and Rb, for proteosomal degradation. The method provided by the present invention inhibits the growth of a tumor cell by administering to the cell an effective amount of p19 ARF  or a mimetic thereof, and p53 to inhibit the growth of the tumor cell. Also provided by the present invention are pharmaceutical composition comprising p19 ARF  or a mimetic thereof, and/or p53.

STATEMENT OF GOVERNMENT INTEREST

[0001] This invention was made with government support under NIH GrantNo. RO1EY09300. As such, the government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0002] Growth control in mammalian cells is accomplished largely by theRb protein regulating exit from the GI phase (Weinberg, 1995, Cell 81,323-330) and the p53 protein triggering growth arrest/apoptoticprocesses in response to cellular stress (Levine, 1997, Cell 88,323-331). Cross-talk between these two regulatory pathways may bemediated through the p21 cdk inhibitor, which is a target of p53transactivation as well as a factor that influences the functionalstatus of Rb (Weinberg, 1995, Cell 81, 323-330.). An additional level ofoverlap between p53 and Rb is provided by the MDM2 protein that canphysically associate with both proteins and prevent their growthsuppression (Momand et al., 1992, Cell 69, 1237-1245; Xiao et al., 1995,Nature 375, 694-698). In tumorigenesis RB and p53 appear to servecollaborative roles as evidenced by the observations that many tumortypes exhibit mutations in both RB and p53 (Williams et al., 1994,Nature Genet. 7, 480-484) and mice that are RB+/− and p53−/− develop awider range of tumors at earlier ages than mice that are either Rb+/− orp53−/− (Williams et al., 1994, Nature Genet. 7, 480-484). Moreover, theability of several viruses to transform cells in culture and causetumors in mice is due to viral oncoproteins that bind to and inactivateboth RB and p53 (Hawley-Nelson et al., 1989, EMBO 8, 3905-3910; Mungeret al., 1989, Journal of Virology 63, 4417-4421; Mahon et al., 1987,Science 235, 1622-1628; Symonds et al., 1994, Cell 78, 703-711). Themechanistic basis for this dual requirement stems in part from thedeactivation of a p53-dependent cell suicide program that would normallybe brought about as a response to unchecked cellular proliferationresulting from Rb-deficiency (Ko and Prives, 1996, Genes Devel. 10,1054-1072; Gottlieb and Oren, 1996, Biophysica Acta. 1287, 77-102;Levine, 1997, Cell 88, 323-331).

[0003] p53 mutation is thought to be the most frequent geneticalteration in human cancers (Hollstein et al., 1991, Science 253, 49-53;Levine et al., 1991, Nature 351, 453-456). In proliferating normal andneoplastic cells, the consequences of p53 over-expression arecontext-dependent, resulting in either cell cycle arrest or induction ofapoptosis (Ko and Prives, 1996, Genes Devel. 10, 1054-1072). Thesebiological endpoints provide a basis for p53's anti-oncogenic actions(Eliyahu et al., 1989, Proc. Natl. Acad. Sci. USA 86, 8763-8767; Finlayet al., 1989, Cell 57, 1083-1093) and have been shown to relate to itscapacity to function as a sequence-specific transcription factor(Pietenpol et al., 1994, Proc. Natl. Acad. Sci. USA 91, 1998-2002; Crooket al., 1994, Cell 79, 817-827), and to interact with key cellularproteins. The critical role served by p53 in these diverse physiologicalprocesses necessitates that p53 activity be subject to stringentmulti-level regulation. One crucial level of regulation involves theMDM2 protein whose direct interaction with p53 blocks p53-mediatedtransactivation (Chen et al., 1995, Mol. Med 1, 141-142) and targets thep53 protein for rapid degradation (Levine, 1997, Cell 88, 323-331;Kubbutat et al., 1997, Nature 387, 299-303; Haupt et al., 1997, Nature387, 296-299). MDM2 itself has been shown to be amplified in primarytumors (Oliner et al., 1992, Nature 362, 857-860), to act as animmortalizing oncogene in cell culture (Finlay, 1993, Molecular &Cellular Biology 13, 301-306), and to directly repress basaltranscription (Thut et al., 1997, Genes Devel. 11, 1974-1986).

[0004] In human cancers, disruption of the RB pathway can result frominactivation of RB itself through gene mutation/deletion, viralsequestration or hyperphosphorylation (Weinberg, 1995,Cell 81, 323-330),or through disregulation of the components controlling the degree of RBphosphorylation. The latter can take place through activating mutationsin the G1 specific Cyclin-Dependent Kinase 4 (CDK4) catalytic unit,up-regulation of D-type cyclin levels, and/or elimination of INK4s (forINhibitors of Cyclin-Dependent Kinase 4) (Sherr, 1996, Science 274,1672-1676). The products of INK4 family genes have been shown to bind toCDK4 and inhibit CDK4-directed phosphorylation of Rb (Quelle, et al.,1995a, Oncogene 11, 635-645; Serrano et al., 1993, Nature 366, 704-707),thereby blocking exit from the G1 phase of the cell cycle (Sherr, 1996,Science 274, 1672-1676). One member of the INK4 family, INK4a, has beenshown to exhibit loss of function in a wide spectrum of tumor types;this pathogenetic event appears to be exceeded in frequency only by p53inactivation. The basis for the prominence of INK4a, as opposed to othermembers of the INK4 family, in tumor suppression is not fully understoodbut may relate to its unusual capacity to encode two distinctproteins—the cyclin-dependent kinase inhibitor, p16^(INK4a), and a novelprotein of unknown function, p19^(ARF). This special feature of INK4aresults from a unique gene organization in which the two INK4a geneproducts are encoded by different first exons and alternative readingframes residing in a common second exon. The fact that both geneproducts are often eliminated or mutated in many cancers has raisedquestions regarding their relative contributions to INK4a-mediated tumorsuppression.

[0005] Compelling support for p16^(INK4a) as a critical target oftumorigenesis includes germline mutations/deletions exclusivelyaffecting the p16^(INK4a) ORF in melanoma-prone kindreds and atumor-associated CDK4 mutation rendering this kinase insensitive top16^(INK4a) inhibition (Zuo et al., 1996, Nature Genet. 12, 97-99). Withregard to p19^(ARF), although direct evidence linking loss of p19^(ARF)function with human tumorigenesis has been lacking, many INK4amutations/deletions map to the exon 2 region that is shared by p19^(ARF)and a p19^(ARF)-specific knockout leads to spontaneous tumor formationin mice (Kamijo et al., 1997a, Cell 91, 649-659).

[0006] Some clues addressing p19^(ARF)'s mechanism of action have beenprovided by the requirement for p53 in p19ARF-induced G1 arrest and byan absence of p53 mutations in post-crisis p19^(ARF)−/− MEF cultures(Kamijo et al., 1997a, Cell 91, 649-659) and in RAS-induced melanomasarising in the INK4a null mice (Chin et al., 1997, Genes and Development11, 2822-2834). Additionally, studies reported here suggest thatp19^(ARF) requires p53 function to suppress cellular transformation. Allof these observations have led to the intriguing possibility that theINK4a gene is linked not only to the Rb pathway through p16^(INK4a) butalso to the p53 pathway through p19^(ARF).

[0007] The present invention describes the determination of the functionof the novel protein p19^(ARF). The inventors have determined that thenovel p19^(ARF) protein acts as a suppressor of oncogenictransformation. The inventors specifically ascertained that p19^(ARF)engages the p53 pathway through physical interactions with the MDM2oncoprotein. p19^(ARF) specifically inhibits the oncogenic actions ofMDM2, blocks MDM2-induced degradation of p53, and enhances p53-dependenttransactivation. The inventors additionally demonstrated that loss ofINK4a attenuates apoptosis brought about by Rb deficiency. These studiesprovide physical and mechanistic insight fortifying INK4a's position atthe nexus of the two most important tumor suppressor pathways governingthe development of neoplasia, and provide an explanation for thefrequent involvement of INK4a in tumorigenesis.

SUMMARY OF THE INVENTION

[0008] The present invention provides a novel method of inhibiting thegrowth of tumor cells based upon the discovery that p19^(ARF) acts as asuppressor of oncogenic transformation by binding to the MDM2oncoprotein and blocking MDM2's ability to target associated proteins,such as p53 and Rb, for proteosomal degradation.

[0009] The present invention specifically provides a method ofinhibiting the growth of a tumor cell by introducing to the cell aneffective amount of p19^(ARF) or a mimetic thereof, and p53 to inhibitthe growth of the tumor cell.

[0010] The present invention further provides a pharmaceuticalcomposition comprising p19^(ARF) in the form of a protein, a nucleicacid encoding p19^(ARF), a nucleic acid encoding p19^(ARF) contained ina vector, or a mimetic thereof. Also provided is a pharmaceuticalcomposition comprising p19^(ARF) in the form of a protein, a nucleicacid encoding p19^(ARF), a nucleic acid encoding p19^(ARF) contained ina vector, or a mimetic thereof, and p53.

[0011] Additional objects of the present invention will be apparent fromthe description which follows.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIGS. 1A and 1B. FIGS. 1A and 1B set forth an anti-oncogenicprofile of the mouse p16^(Ink4a) and p19^(ARF) expression constructs inMyc/RAS versus E1a/RAS cotransformation assays. FIG. 1A shows ahistogram of a representative REF cotransformation assay showing theaverage number of foci per 10 cm plate following cotransfections withmouse c-myc, H-RAS^(val12) and 2 μg of the various expression constructslisted above each bar. FIG. 1B shows the same experimental design asdescribed in FIG. 1A except that each plate was cotransfected with 2 μgE1a, H-RAS^(val12), and the various expression constructs listed. Inthis particular experiment, the p16^(Ink4a) transfection point exhibitedan artificially low number of foci relative to the empty vector.Although this decline is not statistically significant, in all otherexperiments the addition of human or mouse p16^(Ink4a) has little or noinhibition against E1a/RAS transformation, similar to previous studies(Serrano M., et al., Science 267, 249-252 (1995)). Support for the lackof an effect also derives from the lack of additional suppression byp16^(Ink4a) in the p16^(Ink4a)+p19^(ARF) transfection point compared top19^(ARF) alone. The general cyclin-dependent kinase inhibitorp21^(CIP1) served as a positive control for an inhibitory agent actingin G1 and G2M phases of the cell cycle.

[0013] FIGS. 2A-2C. FIGS. 2A-2C demonstrate the anti-oncogenic activityof p19^(ARF) in relation to p53. FIG. 2A sets forth a histogram of arepresentative REF cotransformation assay showing the average number offoci per 10 cm plate following cotransfections with 2 μg each of mouseTag, H-RAS^(val12) and empty vector or p19^(ARF). FIG. 2B is similar toA except that p53KH213 and H-RAS^(val12) were used to generate foci.FIG. 2C shows a Myc/RAS cotransformation assay conducted with MEFshomozygous null for p53. The bars represent the % foci generated in thepresence of 2 μg p16^(INK4a) or p19^(ARF) relative to control platesreceiving 2 μg c-myc, 2 μg RAS and 2 μg empty vector. In the MEF assays,clear countable Myc/RAS foci are not generated in wildtype MEF cultures,thus a direct comparison to MEFs wildtype for p53 is not possibleHowever, INK4a−/−MEF cultures that are wildtype for p53 and do allow forreadable foci counts (Serrano, M., et al., Cell 85, 27-37 (1996))exhibited a 4-fold reduction with the addition of p19^(ARF) to Myc/RAScotransfections (data not shown), a result comparable to that obtainedwith REF cultures.

[0014]FIGS. 3A and 3B. FIGS. 3A and 3B represent the P19^(ARF)-MDM2interaction in vivo. FIG. 3A shows an IP-Western analysis of 293T cellstransfected with the indicated expression constructs above the panel.The α-MDM2 (lanes 2 and 3) and α-FLAG (lanes 4-6) immunoprecipitateswere Western-blotted and assayed with an α-MDM2 antibody. FIG. 3B setsforth an IP-Wextern analysis of untransfected 3T3DM employing theantibody listed below the panel (NI, nonimmune; H.C., heavy chain) andblotted with α-MDM2 antibody. FIG. 3C shows a confocal microscopicanalysis of MDM2 and p19^(ARF) protein distribution in 293T and 3T3DMcells. The yellow signal indicates co-localization of the two proteins.A staining pattern similar to that observed for p19^(Flag) in 293T cellshas been observed previously for endogenous p19^(ARF) (Quelle D E., etal., Cell 83, 993-1000 (1995)), indicating that the nucleolarlocalization in 293Ts is not an artifact of over-expression or epitopetagging.

[0015] FIGS. 4A-4I. FIGS. 4A-4I demonstrate the effect of p19^(ARF) onp53-mediated apoptosis and transactivation. Representative sections ofage-matched lenses showing morphology by H&E stain, proliferation byBrdU incorporation, and apoptosis by TUNEL assay in E14.5 wildtype (FIG.4A, FIG. 4D and FIG. 4G), Rb−/− (FIG. 4B, FIG. 4E and FIG. 4H) andRb−/−, INK4a−/− (FIG. 4C, FIG. 4F and FIG. 4I) lenses are shown. Thelenses are oriented with the anterior epithelium (ep) facing the lowerleft corner and the lens fiber region (lf) facing the upper rightcorner. The arrows in FIG. 4H and FIG. 4I point to TUNEL-positivenuclei.

[0016]FIGS. 5A and 5B. FIG. 5A shows a quantitative comparison ofapoptosis nuclei relative to the total number of lens fiber nuclei inE13.5 and E14.5 Rb-1- (solid) and Rb−/−, INK4a−/− lenses (striped). FIG.5B shows a CAT reporter quantitation following transfection of theindicated plasmids containing the CAT gene driven by p53-responsive(PG13) or non-responsive (MG13) promoters. Each transfection pointreceived wildtype p53 and the indicated amount of p19^(ARF) construct.Basal activity of the PG13 CAT reporter was arbitrarily assigned a valueof 1.0.

[0017] FIGS. 6A-6D. FIG. 6A demonstrates the cooperative effects of themouse p16^(Ink4a) and p19^(ARF) expression constructs in Myc/RAScotransformation assays. Histogram of a representative REFcotransformation assay showing the average number of foci per 10 cmplate following cotransfections with 2 μg mouse c-myc, H-RAS^(val12) andthe various expression constructs listed above the error bars. FIG. 6Bsets forth the distinct actions of p16^(Ink4a) and p19^(ARF) expressionconstructs in E1a/RAS cotransformation assays. The same experimentaldesign as described in FIG. 6A except that each plate was co-transfectedwith 2 μg E1a, H-RAS^(val12), and the various expression constructslisted. In this particular experiment, the p16^(Ink4a) transfectionpoint exhibited an usually low number of foci relative to the emptyvector. Although this decline is not statistically significant, in allother experiments the addition of mouse p16^(Ink4a) had no inhibitionagainst E1a/RAS transformation, similar to previous studies with thehuman p16^(Ink4a) (Serrano et al., 1995). Support for the lack of aneffect also derives from the lack of additional suppression byp16^(Ink4a) in the p16^(Ink4a)+p19^(ARF) transfection point compared top19^(ARF) alone. The general cyclin-dependent kinase inhibitorp21^(CIP1) served as a positive control for an inhibitory agent actingdownstream of Rb. FIG. 6C shows anti-oncogenic activity of p19^(ARF) inT-Ag/RAS or dominant negative p53/RAS REF cotransformation assays. Onthe left, histogram of a representative REF cotransformation assayshowing the average number of foci per 10 cm plate followingcotransfections with 2 μg each of T-Ag, H-RAS^(val12) and empty vectoror p19^(ARF). On the right, histogram showing the average number of fociper 10 cm plate following cotransfections with 2 μg each of p53 KH215(encoding a dominant negative mutant p53) and H-RAS^(val12) with orwithout p19^(ARF). FIG. 6D sets forth anti-oncogenic activity ofp19^(ARF) in Myc/RAS MEF cotransformation assays. The early passage MEFsused for each experiment were either null for INK4a (left panel) or nullfor both INK4a and p53 (right panel). The bars represent the number offoci generated in the presence of p19^(ARF) relative to control platesreceiving 2 μg c-myc, 2 μg RAS and 2 μg empty vector. These assays wereperformed on an Ink4a null background because wild-type MEFs do not giveclear, countable foci in Myc/RAS cotransformation assays.

[0018] FIGS. 7A-7F. FIGS. 7A-7F set forth an analysis of the p19ARFcomplex in vivo. FIG. 7A shows a co-immunoprecipitation assay withanti-p53 antibody following transfection of the indicated expressionconstructs into 293T cells. Western blots were probed with anti-FLAGantibody (HC heavy chain, LC light chain) (15% SDS-PAGE). FIG. 7B setsforth 293T cells transfected with the indicated expression constructswere metabolically labeled, and immunoprecipitations using anti-FLAG(lanes 5-8) or anti-MDM-2 (lanes 9-12) antibodies were performed.Precipitated proteins were analyzed on a 4-15% SDS-PAGE gradient gel.FIG. 7C depicts an immunoprecipitation-Western blot analysis of 293Tlysates following transfection with the indicated expression constructs.The lysates were immunoprecipitated with the antibodies indicated belowthe lanes and the blots were probed with an anti-MDM2 antibody (8%SDS-PAGE). FIG. 7D is the same as FIG. 7C except that lysates werederived from untransfected 3T3DM cells which express high levels ofMDM2, p19^(ARF) and p53. The asterisk marks the MDM2 forms that do notinteract with p53. The anti-p19^(ARF) is a rabbit polyclonal p19^(ARF)antisera and is compared with non-immune rabbit serum (NRS) (8%SDS-PAGE). FIG. 7E is the same as FIG. 7C except that SAOS2 cells wereused. FIG. 7F sets forth a confocal microscopic analysis of MDM2 andp19^(ARF) protein distribution in 293T nuclei. The yellow signalindicates co-localization of the two proteins. A staining patternsimilar to that observed for p19^(FLAG) in 293T cells has been observedpreviously for p19^(ARF) (Quelle et al., 1995b, Cell 83, 993-1000),supporting that this apparent nucleolar localization pattern is not anartifact of over-expression or epitope tagging.

[0019]FIGS. 8A and 8B. FIGS. 8A and 8B show the localization ofp19^(ARF) interaction region of MDM2. FIG. 8A sets forth a graphicrepresentation of the full length MDM2 protein (top) and MDM2 deletionmutants. The known structural motifs and functional domains of MDM2 areindicated. With regard to the 1-58 mutant, previous studies havedetermined that these sequences are essential for MDM2 interaction withp53 (Oliner et al., 1993, Nature 362, 857-860; Momand et al., 1992, Cell69, 1237-1245; Kussie et al., 1996, Science 274, 948-953). FIG. 8B setsforth a western blot analysis of lysates (lanes 2,4,6,8,10,12,14) and ofanti-FLAG immunoprecipitates (lanes 1,3,5,7,9,11,13,15) followingtransient transfection of the indicated expression constructs into 293Tcells (lanes 1-7, 10-13) or SAOS2 cells (lanes 8-9, 14-15). Asterisksdenote the MDM2 band of interest in each immunoprecipitate. Lanescontaining lysate demonstrate that the transfected MDM2 and its mutantderivatives are expressed at high levels. Note the absence of an MDM2band in the anti-FLAG precipitates after transfection of 155-491 andp19^(FLAG). The nuclear localization of each mutant protein wasconfirmed by in situ immunohistochemistry (data not shown). The Westernblots were probed with anti-MDM2 monoclonal antibody directed to anepitope (aa 26-168) present within all of the MDM2 proteins used in thisassay. 8% SDS-PAGE (lanes 1-9). 14% SDS-PAGE (lanes 10-15). HC, heavychain. LC, light chain.

[0020] FIGS. 9A-9D. FIGS. 9A-9D show the effect of p19^(ARF) onMDM2-related functions. FIG. 9A sets forth a representative MDM2/RAScotransformation experiment comparing transformed foci counts inMDM2/RAS cotransfections receiving either empty vector, p16^(INK4a) orp19^(ARF). FIG. 9B: Top panel: Western blot analysis of HeLa celllysates probed with anti-p53 antibody (Ab-1 Calbiochem) followingtransfection of the indicated expression constructs. Middle panel:Western blot of the same lysates probed with an anti-MDM2 antibody. Noteinduction of endogenous MDM2 upon transfection of p53. The very modestreduction in MDM2 levels observed upon addition of p19^(ARF) (lanes 3versus 4) is not likely to account for the p19^(ARF) effect since MDM2levels are greatly increased over those observed in the p53 alonetransfections (compare lanes 4 and 2). Bottom panel: Western blot probedwith anti-FLAG antibody showing a non-specific cross reacting FLAGepitope (NSFE) used as a loading control. FIG. 9C shows the highermolecular weight forms of p53 that are induced by MDM2 and thought torepresent polyubiquitinated p53 targeted for proteasomal degradation.Note that there is a decrease in these bands upon addition of p19^(ARF).In this particular experiment, visualization of these p53 bands isfacilitated by transfection of higher amounts of p53, use of 2 differentanti-p53 antibodies (DO-1 and 1801), and film over-exposure. FIG. 9D:Top panel: p53-dependent CAT reporter assays documenting the effects ofp19^(ARF), MDM2 or both on p53 transactivation activity. Amounts loadedare normalized for transfection efficiency. For these SAOS2transfections, the amounts of DNA used were either 0.2 or 0.5 μg for p53and 2 μg each for MDM2 or p19^(ARF). Bottom panel: Histogramrepresentation of p53 CAT activities as determined by PhosphorImagerquantitation of signal intensities.

[0021]FIGS. 10A and 10B. FIGS. 10A and 10B show the effect of INK4adeficiency on proliferation and apoptosis in the Rb-deficient lens invivo. FIG. 10A sets forth representative sections of age-matched lensesshowing morphology by H&E stain, proliferation by BrdU incorporation,and apoptosis by TUNEL assay in E14.5 wildtype (a,d,g), Rb−/− (b,e,h)and Rb−/−, INK4a−/− (c,f,i) lenses. The lenses are oriented with theanterior epithelium facing the lower left corner and the lens fiberregion facing the upper right corner. TUNEL-positive nuclei are stainedbrown by HRP reaction. FIG. 10B: Left Panel: Quantitative comparison ofS phase nuclei (BrdU-positive) relative to the total number of lensfiber nuclei in E13.5 and E14.5 Rb−/− (solid) and Rb−/−, INK4a−/− lenses(striped) (data compiled from examining 332 sections from 19 embryos).Right Panel: Quantitative comparison of apoptotic nuclei(TUNEL-positive) relative to the total number of lens fiber nuclei inE13.5 and E14.5 Rb−/− (solid) and Rb−/−, INK4a−/− lenses (striped) (datacompiled from examining 174 sections from 13 embryos).

[0022]FIGS. 11A and 11B. FIG. 11A sets forth a schematic of the Onegene-two products-two pathways hypothesis positioning the INK4a genealong the Rb and p53 tumor suppressor pathways. FIG. 11B sets forth theproposed mechanism for p19^(ARF)'s enhancement of p53-related functions.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides a novel method of inhibiting thegrowth of tumor cells. This method is based upon the important discoverythat p19^(ARF) acts as a suppressor of oncogenic transformation bybinding to the MDM2 oncoprotein and blocking MDM2's ability to targetassociated proteins, such as p53 and Rb, for proteosomal degradation.

[0024] The present invention specifically provides a method ofinhibiting the growth of a tumor cell by introducing to the cell aneffective amount of p19^(ARF) or a mimetic thereof, and p53 to inhibitthe growth of the tumor cell.

[0025] The p19^(ARF) and p53 proteins of the present invention may bethe wild type proteins, or analogues thereof, and may be producedsynthetically or recombinantly, or may be isolated from native cells. Asused herein, “analogue” means functional variants of the wild typeprotein, and includes p19^(ARF) or p53 protein isolated from mammaliansources other than human, as well as functional variants thereof.

[0026] As used herein, “mimetic” is any agent, such as a syntheticchemical or a protein, which, like p19^(ARF), binds to MDM2 and blocksMDM2's ability to target an associated protein, such as p53 and Rb, forproteosomal degradation. In a specific embodiment of the invention, themimetic prevents MDM2's ability to target p53, thereby blockingdegradation of p53. p19^(ARF)-like mimetics may be sterochemicallydesigned so that, based upon the protein structure and characteristicsof p19^(ARF), they will bind to the MDM2 protein and prevent its abilityto target proteins.

[0027] Alternatively, the mimetics provided by the present invention maybe nucleic acid sequences or proteins that directly bind to the aminoacid sequence or the nucleic acid sequence of the MDM2 protein andthereby effect transcription or translation of the sequence, ultimatelyinhibiting or altering the ability of the MDM2 protein to targetassociated proteins, such as p53 and Rb, for proteosomal degradation.The sequences of these nucleic acids and proteins may be determinedbased upon the amino acid sequence or the nucleic acid sequence of MDM2and its area of interaction with p19^(ARF).

[0028] The mimetics of the present invention may comprise proteins,polypeptides, peptides, nucleic acid sequences, and small non-peptideorganic molecules that have been shown to inhibit the interaction ofMDM2 with p53. The nucleic acid sequences may comprise RNA, antisenseRNA, double stranded RNA, RNA-DNA hybrids, double stranded DNA,nucleotides, oligonucleotides, or antisense oligonucleotides that alsodirectly bind to or alter the ability of MDM2 to target associatedproteins involved in proteosomal degradation.

[0029] Once the sequence of the mimetic is determined, the sequences maythen be prepared in several ways. For example, the nucleotide sequencesmay be isolated from a natural source, or may be synthesized usingrecombinant DNA techniques. The amino acid sequences may be synthesizedby methods commonly known to one skilled in the art (Modern Techniquesof Peptide and Amino Acid Analysis, John Wiley & Sons (1981); M.Bodansky, Principles of Peptide Synthesis, Springer Verlag (1984)).Examples of methods that may be employed in the synthesis of the aminoacids sequences, include, but are not limited to, solid phase peptidesynthesis, solution method peptide synthesis, and synthesis using any ofthe commercially available peptide synthesizers. The amino acidsequences may contain coupling agents and protecting groups used in thesynthesis of the protein sequences, and are well known to one of skillin the art.

[0030] p19^(ARF) or a mimetic thereof, and p53 may be introduced to acell as a protein, nucleic acid, or nucleic acid contained in a vector.It is to be understood that any combination of these may be used. Forexample, p19^(ARF) or a mimetic thereof may be administered to a cell asa protein while p53 is administered to the cell as nucleic acid encodingp53, or nucleic acid encoding p53 contained in an expression vector. Inaddition, nucleic acid encoding p19^(ARF) or a mimetic thereof, ornucleic acid encoding p19^(ARF) or a mimetic thereof contained in anexpression vector may be administered to a cell, while p53 isadministered to the cell as a protein.

[0031] Nucleic acid encoding p19^(ARF) or a mimetic thereof, and p53 andnucleic acid encoding p19^(ARF) or a mimetic thereof, and p53 containedin an expression vector, may be administered to a tumor cell using manymethods known to one skilled in the art. For example, nucleic acidencoding p19^(ARF) or a mimetic thereof, and p53 contained in anexpression vector may be introduced to a cell using gene therapy. Genetherapy comprises the introduction of a recombinant vector containing anucleic acid sequence encoding p19^(ARF), a mimetic thereof, or p53 intoa cell. The recombinant vector containing DNA encoding p19^(ARF), amimetic thereof, or p53, may be introduced into the tumor cell using anynumber of procedures known to one skilled in the art, such aselectroporation, DEAE Dextran, monocationic liposome fusion,polycationic liposome fusion, protoplast fusion, DNA coatedmicroprojectile bombardment, by creation of an in vivo electrical field,injection with recombinant replication-defective viruses, homologousrecombination, and naked DNA transfer. It is to be appreciated by oneskilled in the art that any of the above methods of nucleic acidtransfer may be combined. Accordingly, a stem cell which expressesp19^(ARF) or a mimetic thereof, and p53 introduced therein through viraltransduction, homologous recombination, or transfection is also providedby the present invention.

[0032] The recombinant vector may comprise a nucleic acid of orcorresponding to at least a portion of the genome of a virus, where thisportion is capable of directing the expression of a DNA sequence, andthe nucleic sequence encoding a p19^(ARF) or a mimetic thereof, or p53,operably linked to the viral nucleic acid and capable of being expressedas a functional gene product in the target tumor cell. The recombinantvectors may be derived from a variety of viral nucleic acids known toone skilled in the art, e.g. the genomes of HSV, adenovirus,adeno-associated virus, Semiliki Forest virus, vaccinia virus, and otherviruses, including RNA and DNA viruses.

[0033] The recombinant vectors may also contain a nucleotide sequenceencoding suitable regulatory elements so as to effect expression of thevector construct in a suitable host cell. As used herein, “expression”refers to the ability of the vector to transcribe the inserted DNAsequence into mRNA so that synthesis of the protein encoded by theinserted nucleic acid can occur. Those skilled in the art willappreciate that a variety of enhancers and promoters are suitable foruse in the constructs of the invention, and that the constructs willcontain the necessary start, termination, and control sequences forproper transcription and processing of the nucleic acid sequenceencoding a protein involved in the regulation of smooth muscle tone whenthe recombinant vector construct is introduced into a host cell.

[0034] Vectors suitable for the expression of the nucleic sequenceencoding p19^(ARF) or a mimetic thereof, or p53 are well known to oneskilled in the art and include pET-3d (Novagen), pProEx-1 (LifeTechnologies), pFastBac 1 (Life Technologies), pSFV (Life Technologies),pcDNA II (Invitrogen), pSL301 (Invitrogen), pSE280 (Invitrogen), pSE380(Invitrogen), pSE420 (Invitrogen), pTrcHis A,B,C (Invitrogen), pRSETA,B,C (Invitrogen), pYES2 (Invitrogen), pAC360 (Invitrogen), pVL1392 andpVl1392 (Invitrogen), pCDM8 (Invitrogen), pcDNA I (Invitrogen), pcDNA I(amp) (Invitrogen), pZeoSV (Invitrogen), pcDNA 3 (Invitrogen), pRc/CMV(Invitrogen), pRc/RSV (Invitrogen), pREP4 (Invitrogen), pREP7(Invitrogen), PREP8 (Invitrogen), pREP9 (Invitrogen), pREP10(Invitrogen), pCEP4 (Invitrogen), pEBVHis (Invitrogen), and λPop6. Othervectors would be apparent to one skilled in the art.

[0035] Suitable promoters include, but are not limited to, constitutivepromoters, tissue specific promoters, and inducible promoters.Expression of the nucleic acid sequence encoding p19^(ARF) or a mimeticthereof, or p53 may be controlled and affected by the particular vectorinto which the nucleic acid sequence has been introduced. Someeukaryotic vectors have been engineered so that they are capable ofexpressing inserted nucleic acids to high levels within the target cell.Such vectors utilize one of a number of powerful promoters to direct thehigh level of expression. Eukaryotic vectors use promoter-enhancersequences of viral genes, especially those of tumor viruses. Thisparticular embodiment of the invention provides for regulation ofexpression of the nucleic acid sequence encoding p19^(ARF) or a mimeticthereof, or p53 using inducible promoters. Non-limiting examples ofinducible promoters include, but are not limited to, metallothioninepromoters and mouse mammary tumor virus promoters. Depending on thevector, expression of the nucleic acid sequence encoding p19^(ARF) or amimetic thereof, or p53 would be induced in the tumor cell by theaddition of a specific compound at a certain point in the growth cycleof the cell. Other examples of promoters and enhancers effective for usein the recombinant vectors include, but are not limited to, CMV(cytomegalovirus), SV40 (simian virus 40), HSV (herpes simplex virus),EBV (epstein-barr virus), retroviral, adenoviral promoters andenhancers, and tumor cell specific promoters and enhancers.

[0036] Also provided by the present invention are pharmaceuticalcompositions comprising the p19^(ARF) protein, DNA encoding p19^(ARF),or a recombinant vector encoding p19^(ARF), together with apharmaceutically or physiologically acceptable carrier. Thepharmaceutical composition may additionally contain p53 in the form of aprotein, nucleic acid expressing p53, or nucleic acid expressing p53contained in a vector. The pharmaceutical composition may furthercomprise p16^(Ink4a) in the form of a protein, nucleic acid expressingp16^(Ink4a), or nucleic acid expressing p16^(Ink4a) contained in avector. Such compositions may be prepared by adding the p19^(ARF)protein, nucleic acid encoding p19^(ARF), or a recombinant vectorencoding p19^(ARF) separately or in combination with p53 protein,nucleic acid encoding p53, or a recombinant vector encoding p53 to thepharmaceutically or physiologically acceptable carrier. Suitablepharmaceutically or physiologically acceptable carriers include, but arenot limited to, water containing physiologically compatible substancessuch as sodium chloride, glycine, and the like, having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile.

[0037] Further provided by the present invention is a method of treatingcancer by preventing the occurrence of and inhibiting the progression ofmany different types of cancers and benign proliferation disorders byadministering to a subject an effective amount of p19^(ARF), or amimetic thereof, and p53 to inhibit the growth of the cancer.Non-limiting examples of cancers that can be treated using the agentsprovided by the present invention include melanoma, bladder carcinoma,oral carcinoma, lung carcinoma, and lymphoid neoplasms such as B-cellchronic lymphocytic leukemia, Hodgkin's and non-Hodgkin's lymphomas.Non-limiting types of benign proliferation disorders that arecharacterized by an abnormal proliferation of cells include benignnephrosclerosis, benign prostatic hyperplasia. Veterinary uses are alsointended to be encompassed by this invention.

[0038] Administration of the pharmaceutical composition containingp19^(ARF), nucleic acid encoding p19^(ARF), or a mimetic thereof, alongwith p53, may be administered to a tumor cell of a subject by methodsnot limited to gene therapy, creation of an in vivo electrical field,injection with recombinant replication-defective viruses, homologousrecombination, and naked DNA transfer. It is to be appreciated by oneskilled in the art that any of the above methods of DNA transfer may becombined. Accordingly, a stem cell which expresses p19^(ARF) or amimetic thereof, or p53 introduced therein through viral transduction,homologous recombination, or transfection is also provided by thepresent invention may also be administered to a subject to inhibitgrowth of cells.

[0039] Administration of the pharmaceutical composition may be foreither a prophylactic or therapeutic use. When providedprophylactically, the composition is provided in advance to the symptomcaused by the condition afflicting the individual. When providedtherapeutically, the composition is provided at, or shortly after, theonset of any symptoms of the disease. The therapeutic administration ofthe composition serves to attenuate the condition.

[0040] The pharmaceutical composition containing p19^(ARF) protein,nucleic acid encoding p19^(ARF), or nucleic acid encoding p19^(ARF)contained in a vector, or a mimetic thereof, may be administered to asubject, tumor, or cell prior to, simultaneously with or subsequent toadministration of p53 protein, nucleic acid encoding p53 protein, ornucleic acid encoding p53 protein contained in a vector.

[0041] For the purposes of gene transfer into a tumor cell of a subject,a recombinant vector containing nucleic acid encoding p19^(ARF) or amimetic thereof, or p53 may be combined with a sterile aqueous solutionwhich is preferably isotonic with the blood of the recipient. Suchformulations may be prepared by suspending the recombinant vector inwater containing physiologically compatible substances such as sodiumchloride, glycine, and the like, and having buffered pH compatible withphysiological conditions to produce an aqueous solution, and renderingsuch solution sterile. In a preferred embodiment of the invention, therecombinant vector is combined with a 20-25% sucrose in saline solutionin preparation for introduction into a smooth muscle cell.

[0042] The amounts of p19^(ARF), nucleic acid encoding p19^(ARF), ornucleic acid encoding p19^(ARF) contained in a vector, or a mimeticthereof, and p53 are in amounts sufficient to inhibit the growth of atumor cell. However, the exact dosage will depend on such factors as thepurpose of administration, the mode of administration, the size of thetumor or cancerous tissue, the stage of progression of the disease, andthe efficacy of the composition, as well as the individualpharmacokinetic parameters of the subject. One of skill in the art willknow the parameters to evaluate the response of the individual to thecomposition containing and establish the dosage based on thoseparameters. Such therapies may be administered as often as necessary andfor the period of time as judged necessary by the treating physician.

[0043] The present invention is described in the following ExperimentalDetails Section which is set forth to aid in the understanding of theinvention, and should not be construed to limit in any way the scope ofthe invention as defined in the claims which follow thereafter.

[0044] Experimental Details Section

[0045] I. Materials and Methods

[0046] Expression Constructs and REF Cooperation Assays. Expressionconstructs encoding mouse p16^(Ink4a) and p19^(ARF) were generated byplacing the complete ORFs derived from their respective cDNAs(Schreiber-Agus et al., 1994, Oncogene 9, 3167-3177) in the senseorientation relative to two tandemly repeated Moloney murine leukemiavirus (MuLV) long terminal repeats in the pVNic vector (Schreiber-Aguset al., 1997, Cell 1995. March 10. 80, 777-786). Expression constructsfor c-myc, mutant H-RAS, and E1a have all been described previously(Schreiber-Agus et al., 1997, Cell 1995. March 10. 80, 777-786) and theCMV-driven expression construct encoding the KH215 dominant-negativemutant form of p53 (Gruis et al., 1995, Am. J. Pathol. 146, 1199-1206)has been described previously. To perform the rat embryo fibroblast(REF) cooperation assays, early passage cultures of REFs were preparedand cotransfected as described previously (Schreiber-Agus et al., 1997,Cell 1995. March 10. 80, 777-786) with DNA mixtures containing 2 μg eachof the relevant expression constructs plus the corresponding amount ofcarrier DNA, for a total of 30 μg DNA. At 9 to 12 dayspost-transfection, foci were scored visually and confirmed bymicroscopic examination to be transformed morphologically. For the MEFassays, early passage MEFs were prepared from day 13.5 embryos mincedand seeded into 10 cm plates. The following day cells were split 1:3 andfrozen upon reaching confluency (˜24 hours). MEFs were thawed andtransfected according to the REF assay protocol.

[0047] Protein analysis. For confocal analysis, 293T or 3T3DM cells wereseeded on gelatin coated glass cover slips at a density of 130,000 cellsper 2 cm well. Twenty 24 hrs later, the cells were transfected with 3 μgeach of FLAG-tagged p19^(ARF) and human MDM-2 expression constructsusing Lipofectamine (Gibco BRL). Forty-eight hours post transfection,cells were fixed in 2% paraformaldehyde for 10 min, washed in PBS,permeablized in 1% Triton X-100 for 10 min, blocked with 3% milk in PBSfor 30 min, and incubated in primary antibody diluted in blockingsolution overnight at 4° C. For FLAG-tagged p19^(ARF), M2 antibody(Kodak) was used at a concentration of 5 μg/ml. Anti-MDM-2 Ab-1(Calbiochem) was diluted 1:10. Following this incubation, cells werewashed in PBS and incubated in secondary Ab for 1 hour. The secondaryantibodies (Southern Biotechnology) were anti-lgG2a-Texas Red for FLAGand anti-lgG1-FITC for MDM2. All incubations except for primary antibodywere at RT. Finally, cells were washed, and cover slips were mounted in1:1 glycerol: PBS for viewing on a Bio-Rad MR600 laser scanning confocalmicroscope.

[0048] For immunoprecipitations, subconfluent 293T cells weretransfected with 3 μg each of the appropriate expression constructsshown in FIG. 3, and 80 μg of Lipofectamine reagent (Gibco BRL)Immunoprecipitations under low-stringency conditions were performed(Schreiber-Agus N., et al., Cell 80, 777-786 (1995)) using anti-FLAG M2(Kodak) and anti-MDM-2 Ab-1 (Calbiochem) antibodies. For p19^(ARF), theFLAG epitope was introduced by PCR. The human-Mdm-2 expression constructwas provided by Arnold Levine (Princeton). Untransfected 3T3-DM cellswere lysed in a low stringency buffer as above. 1.6 mg protein wasimmunoprecipitated using 150 μl anti-MDM2 (2A10) (Olson DC., et al.,Oncogene 8, 2353-2360 (1993)) and protein G or anti-p19^(ARF) (4 μl)with protein A for 1 hour. Western blots were probed with 2A10 1:100 todetect endogenous Mdm-2. The p19ARF antibodies were provided by CharlesSherr (St. Judes).

[0049] For the results set forth in FIGS. 6-10, 293T cells (1.4×10⁶ per10 cm plate in DME supplemented with 10% fetal bovine serum, glutamineand antibiotics) were transfected under serum and antibiotic freeconditions with 3 μg each of the appropriate expression constructs shownin FIGS. 7 and 8, and 80 μg of Lipofectamine reagent (Gibco BRL). ForFIG. 7B, cells were metabolically labeled using the EXPRESS ³⁵Sprotein-labeling mix (Dupont-NEN) for 7 hours before collection.Immunoprecipitations under low-stringency conditions (1% NP-40, 10%glycerol, 10 mM NaF, 50 mM-glycerophosphate, protease inhibitors in PBS)were performed as described previously (Schreiber-Agus et al., 1997)using anti-p53 Ab-6 conjugated beads (Calbiochem), anti-FLAG M2 (Kodak)and anti-HDM-2 Ab-1 (Calbiochem) antibodies. For high-stringencyimmunoprecipitations, RIPA buffer (150 mM NaCl, 1%NP-40, 0.5%deoxycholate, 0.1% SDS, and 50 mM Tris) was used. To construct the FLAGepitope-tagged p19^(ARF) construct, PCR was used to fuse in-frame FLAGepitope sequences at the 3′ end of the p19^(ARF) ORF and thesequence-verified PCR product was cloned into the pcDNA(Invitrogen)expression vector. The CMV driven human MDM2 expression construct(pCHDM1A) was provided by Arnold Levine (Princeton). The MDM2 mutant,156-221, was created by standard PCR and utilization of internalrestriction enzyme sites. Specifically, an XbaI site just 5′ to basesencoding amino acid 155 was ligated in-frame to a sequence-verifiedPCR-generated fragment containing the 5′ engineered XbaI site andbeginning with amino acid residue 221 (the oligomers used are5′-CGCCATCTAGACCGGATCTTGATGCTGGT-3′ and 5′-CGAAGGGCCCAACATCTG-3′). The3′ end of this PCR fragment was fused in-frame with the remainder of theMDM2 ORF via a unique ApaI site. The final ligation was performed in theparental pCHDM1A vector in order to reconstitute the MDM2 ORF minussequences encoding amino acids 156 to 221. SAOS2 cells (5×10⁶ per 10 cmplate in DME supplemented with 10% fetal bovine serum, 5% calf serum,glutamine and antibiotics) were transfected by a modified calciumphosphate method as for the CAT assays (see below), and then processedas for 293Ts under low stringency conditions. Untransfected 3T3DM cellswere lysed in a low stringency buffer as above. 1.6 mg of protein wasimmunoprecipitated using 150 Al anti-MDM-2 (2A10) (Olson et al., 1993)and protein G agarose or anti-p19^(ARF) (4 μl) with protein A agarosefor 1 hour. Western blots were probed with 2A10 1:100 to detectendogenous MDM-2. The anti-p19^(ARF) antibodies were provided by CharlesSherr (St. Judes). For p53 degradation studies, 6×10⁵ HeLa cells orH1299 cells maintained in DME supplemented with 10% fetal calf serum andantibiotics, or 5×10⁶ SAOS2 cells maintained as for CAT assay weretransfected by the calcium phosphate method, with 2 μg pC53SN3 (R.Tjian), 5 μg Mdm2, and/or 5 μg p19^(FLAG) and harvested in RIPA buffer24 hours after transfection. Western blots were probed with anti-p53Ab-1 (FIG. 4B), or a mixture of monoclonal p53 antibodies DO-1 and1801(Santa Cruz) all at 1:100 dilution (FIG. 4C). For confocal analysis,293T cells were seeded on gelatin coated glass cover slips at a densityof 130,000 cells per 2 cm well. Twenty-four hours after seeding, thecells were transfected with FLAG-tagged p19^(ARF) and human MDM-2constructs as above. Forty-eight hours post transfection, cells werefixed in 2% paraformaldehyde for 10 minutes, washed in PBS, permeablizedin 1%Triton X-100 for 10 minutes, blocked with 3% milk in PBS for 30minutes, and incubated in primary antibody diluted in blocking solutionovernight at 4° C. For FLAG-tagged p19^(ARF), M2 antibody, (Kodak) wasused at a concentration of 5 g/ml. Anti MDM-2 Ab-1 (Calbiochem) wasdiluted 1:10. Following this incubation, cells were washed in PBS andincubated in secondary Ab for 1 hour. The secondary antibodies (SouthernBiotechnology) were anti-IgG2a-Texas Red for FLAG and anti-IgG1-FITC forMDM-2. All incubations except for primary antibody were at roomtemperature. Finally, cells were washed, and cover slips were mounted in1:1 glycerol:PBS for viewing on a Bio-RAD MR600 laser scanning confocalmicroscope.

[0050] TUNEL and BrdU assays. TUNEL and BrdU incorporation assays wereperformed as described elsewhere (Morgenbesser et al., 1994) on 3 μMparaffin-embedded lens sections prepared as described elsewhere(Morgenbesser et al., 1994).

[0051] CAT reporter assays. Cultures of 293T cells were grown in DME 10%plus fetal calf serum to 50% confluence in 10 cm (Gottlieb TM., et al.,Biochimica et Biophysica Acta. 1287, 77-102 (1996)) tissue culturedishes and transfected by the lipofectamine (BRL) method according themanufacturer's instructions. Cells were harvested 48 hours posttransfection and CAT reporter activity was assayed by acetylation of¹⁴C-labeled chloramphenicol as reported previously (Gorman C M., et al.,Mol. Cell. Biol. 2, 1044-1051 (1982)) except that the extracts wereincubated at 37° C. for 10 min, the samples were resuspended in 201 ofethyl acetate, and the quantities of protein assayed for CAT wereapproximately 50%g per point. Transfection efficiencies were determinedby addition of 2 μg of human growth hormone plasmid to each transfectionpoint and assaying the media for human growth hormone byradioimmunoassay (Nichols Institute) just before cell lysis. Signal werequantitated using PhosphorQuant software. The LTRp53cG(ala) expressionconstruct encodes the wildtype human p53 protein (Eliyahu D., et al.,Proc. Natl. Acad. Sci. USA 86, 8763-8767 (1989)). The reporterconstructs, detailed elsewhere (Kern S E., et al., Science 256,827-830-(1992)), were the PG13-CAT construct bearing a promoter withmultiple copies of the p53 consensus binding site, and the MG-CAT inwhich these p53 binding sites are replaced by nonspecific sequences.

[0052] Cultures of SAOS2 cells were maintained as above and transfectedby calcium phosphate using the same amounts of DNA as in p53 degradationstudies and immunoprecipitation assays, plus 2.5 g PG13CAT, with DMSOshock, 5 hours after addition of the precipitate (Brown et al., 1993,Mol. Cell. Biol. 13, 6849-6857). Cells were harvested 48 hours postshocking and CAT reporter activity was assayed by acetylation of¹⁴C-labeled chloramphenicol as reported previously (Gorman et al., 1982,Mol. Cell. Biol. 2, 1044-1051) except that the extracts were incubatedat 37° C. for 45 min, the samples were resuspended in 20 μl of ethylacetate, and the quantities of protein assayed for CAT wereapproximately 30 μg per point. Transfection efficiencies were determinedby addition of 2 μg of human growth hormone plasmid to each transfectionpoint and assaying the media for human growth hormone byradioimmunoassay (Nichols Institute) just before cell lysis. Signal wasquantitated using PhosphorQuant software.

[0053] II. Results

[0054] Two preliminary observations made in the INK4a knockout modelsuggested that p19^(ARF) may function as a tumor suppresser protein.First, the re-introduction of either p16_(INK4a) or p19^(ARF) intoINK4a−/−fibrosarcoma cell lines resulted in suppression of tumorigenicgrowth in SCID mice (data not shown). Second, molecular analysis oftumors arising in INK4a+/− mice revealed a high incidence of deletion(rather than mutation) of the remaining functional allele (data nowshown). One interpretation of this homozygous codeletion is that itreflects a more efficient genetic strategy to eliminate both INK4a geneproducts since classically loss of tumor suppressor gene functionpresents with deletion of one allele followed by inactivating pointmutation on the second allele (Serrano, M., et al., Cell 85, 27-37(1996); Cordon-Cardo C., Am. J. Pathol. 147, 545-560 (1995)). Theseobservations, along with the previously reported cell cycle-arrestactivity of p19^(ARF) in addition to p16^(INK4a) (Serrano M., et al.,Science 267, 249-252 (1995); Quelle DE., et al., Cell 83, 993-1000(1995); Koh J., et al., Nature 375, 506-510 (1995)), prompted a detailedassessment of the biological activities of p19^(ARF) in normal andneoplastic cells.

[0055] p19^(ARF) is a potent suppressor of oncogenic transformation ofprimary rodent cells. The anti-oncogenic potency of each ink4a geneproduct was tested in the rat embryo fibroblast (REF) cotransformationassay (Land H., et al., Nature 304, 596-602 (1983)). In this highlyquantitative assay, a comparison of the candidate suppressor's activityagainst various oncogene combinations, such as Myc/RAS, E1a/RAS or SV40Large T Antigen ((TAG)/RAS, can provide a measure of its antioncogenicactivity as well as yield mechanistic insight into how this activityrelates to the Rb and p53 pathways.

[0056] The degree of inhibition of E1a/RAS-versus Myc/RAS-induced fociformation by p19^(ARF), p16^(Ink4a), or both was compared in a parallelseries of cotransfections; the results of a representativecotransfection experiment are shown in FIG. 1. The inventors haveobserved previously that human p16^(Ink4a) can inhibit transformation byc-myc/RAS but not E1a/RAS (Serrano M., et al., Science 267, 249-252(1995)), consistent with the model that p16^(Ink4a) operates upstream ofthe Rb-regulated G1/S transition (Serrano M., et al., Science 267,249-252 (1995); Medema R H., et al., Proc. Natl. Acad. Sci. USA 92,6289-6293 (1995); Lukas J., et al., Nature 375, 503-506 (1995)). Similarto its human counterpart, mouse p16^(Ink4a) induced a 1.7- to 3-foldreduction in foci number when added to c-myc/RAS transfections incomparison to empty vector control (FIG. 1; A and B, p16), but failed tocause a statistically significant decrease in E1a/RAS foci formation.The addition of p19^(ARF) to the cotransfections resulted in asignificant reduction in foci formation by both c-myc/RAS (5-10-fold)and E1a/RAS (4- to 5-fold) (FIG. 1; A and B, p19° F.). Moreover, E1a/RASinhibition by p19^(ARF) was not further augmented by the addition ofp16^(Ink4a), again consistent with the lack of effect of p16^(Ink4a) incells in which Rb has been functionally inactivated (FIG. 1C) (SerranoM., et al., Science 267, 249-252 (1995); Medema R H., et al., Proc.Natl. Acad. Sci. USA 92, 6289-6293 (1995); Lukas J., et al., Nature 375,503-506 (1995)). Finally, a near complete inhibition of the transformingactivity of c-myc/RAS by co-addition of p16^(Ink4a) and p19^(ARF)suggests functional cooperation between proteins with differentmechanisms of action (see below) The anti-oncogenic profile of p19^(ARF)in this assay is reminiscent of that of other cell cycle regulations,such as p53 or p21^(CIP1), that function independent of or downstream toRB (for example see FIG. 1B, p21^(CIP1)).

[0057] Functional p53 is required for full oncogenic suppression byp19^(ARF). p19ARF transcript levels are observed to be up-regulated incell lines in which p53 is mutationally inactivated, MDM2 isoverexpressed, or temperature-sensitive TAg is induced (Quelle D E., etal., Cell 83, 993-1000 (1995); Stone S., et al., Cancer Res. 55,2988-2994 (1995); Mao L., et al., Cancer Res. 55, 2995-2997 (1995).These observations, coupled with the fact that p19^(ARF), like p53, ispurported to act in G1 and G2M (Quelle D E., et al., Cell 83, 993-1000(1995); Quelle DE., et al., Proc. Natl. Acad. Sci. USA 94, 669-673(1997), raised the possibility of a functional connection betweenp19^(ARF) and the p53 pathway. To examine this possible relationshipdirectly, the anti-oncogenic activity of p19^(ARF) was assayed in cellsrendered functionally (TAg or dominant-negative p53) or genetically(p53−/−) deficient for p53.

[0058] In the REF assay, the addition of p19^(ARF) to TAg/RAScontransfections was found to have no effect on the number of focigenerated (FIG. 2A) or on the morphological/growth characteristics ofthese foci (data not shown). These results suggested that p19^(ARF) mayrequire p53 for anti-oncogenic activity and ruled out the possibilitythat p19^(ARF) acts in a nonspecific cytotoxic manner to reduce fociformation in the Myc/RAS and E1a/RAS experiments described above. SinceTAg is known to engage many key pathways beyond p53 (Fanning E., Journalof Virology 66, 1289-1293 (1992); Van Dyke T A., Sem. Cancer Biol. 5,47-60 (1994), the possible functional dependency of p19^(ARF) on p53 wastested on two additional levels. First, p19ARF exhibited no inhibitionof foci generated by cotransfection of a dominant-negative mutant formof p53, p53 KH215, and RAS in the REF assay (FIG. 2B). Second and moredirectly, p19^(ARF)-induced suppression of Myc/RAS foci formation wasgreatly attenuated in early passage p53−/− mouse embryonic fibroblasts(MEFs) (FIG. 2C, reduction of only 25% relative to empty vector comparedto 80 to 95% in p53-competent REF cultures; see legend to FIG. 2). Incontrast, p16^(INK4a) remained fully active in this setting (FIG. 2C).Stated differently, a greater than 3-fold increase in p19^(ARF)suppression is observed in the presence of p53 (compare FIG. 2Cp19^(ARF) with FIG. 1A p19^(ARF)). As such, full oncogenic suppressionby p19^(ARF) requires p53, but p19^(ARF) may also engage p53-independentmechanisms as evidenced by the modest degree of p19^(ARF) suppression(25%) that persists in the absence of p53 (p53−/−MEFs).

[0059] P19^(ARF) interacts with MDM2. Results of the above focus assaysprompted us to assess possible physical interactions between p19^(ARF)and p53 or the MDM2 oncoprotein, a key modulator of p53 activity.Initial studies designed to examine these interactions in vitro provedinconclusive due to the propensity of p19^(ARF) to interactnonspecifically with many different GST fusion proteins, this propertythat likely relates to the unusual amino acid composition of p19^(ARF)(22% arginine (Quelle D E., et al., Cell 83, 993-1000(1995)).Accordingly, low stringency coimmunoprecipitation was used to determinethe nature of p19^(ARF) complexes in mammalian cells. Followingtransfection of MDM2 and FLAG-tagged p19^(ARF) (p19FLAG) into 293Tcells, an anti-FLAG antibody was used to immunoprecipitate p19FLAGcomplexes, and the immunoprecipitate was then immunoblotted and probedwith an anti-MDM2 antibody. As shown in FIG. 3A, abundant MDM2 proteinwas present in the p19FLAG immunoprecipitates (lane 6). MDM2 was notdetected in anti-FLAG immunoprecipitates in the absence of p19FLAGexpression (lane 4).

[0060] Since 293T express high levels of TAG and TAG can associate withMDM2 (Brown DR., et al., Mol. Cell. Biol. 13, 6849-6857 (1993)), theinventors then examined the interaction between endogenous p19^(ARF) andMDM2 in the absence of TAg in untransfected 3T3DM cell lines; thesecells express high levels of p19^(ARF) (Barak Y., et al., Genes andDevelopment 8, 1739-1749 (1994)) and MDM2, the latter due to geneamplification (Cahilly-Snyder L., et al., Som. Cell Mol. Gen. 13,235-244 (1987); Fakharzadeh SS., et al., EMBO J. 10, 1565-1569).Moreover, 3T3DM cells express several different species of MDM2 protein(p90/p85, p76/p74 and, in very low amounts, p57) arising probablythrough alternative processing (Barak Y., et al., Genes and Development8, 1739-1749 (1994); Olson D C., et al., Oncogene 8, 2353-2360 (1993)).Of significance is the fact that the p76/p74 MDM2 species are missingthe N-terminal p53 binding domain (Olson D C., et al., Oncogene 8,2353-2360 (1993)). Employing a p19^(ARF)-specific antibody forimmunoprecipitation, abundant MDM2 was readily detected upon Westernblotting of the immunoprecipitates with anti-MDM2 antibody (lane 8),indicating that p19^(ARF)-MDM2 complexes exist in vivo. The p76/p74species which lacks the p53 binding domains is also present in theimmunoprecipitates indicating that p19^(ARF) can interact with MDM2independent of p53. Independent verification of the p19^(ARF)-MDM2interaction was obtained through confocal microscopic analysis of theintracellular distribution of each protein. As shown in FIG. 3C, MDM2and p19^(ARF) colocalize within the nucleus: in 293T cells transfectedwith p19FLAG colocalization is seen predominantly in nucleoli and to alesser extent in the nucleoplasm; in 3T3DM cells, both proteins arefound primarily in the nucleoplasm with more modest staining in thenucleoli. Although the functional significance of the differentdistribution patterns in 293T and 3T3DM cells is not understood, thecontinued colocalization in the setting of substantial changes inintranuclear distribution further supports the concept that they complexin vivo. Finally, the same co-immunoprecipitation strategy was employedto assess whether p53 is present in p19^(ARF) immunoprecipitates fromuntransfected 3T3DM lysates. p53 protein was readily detected, albeit atlower levels that MDM2 (not shown). In summary, these physical dataserve to complement the functional link between INK4a and the p53pathway and suggest that this link is executed on the level of MDM2.

[0061] Functional relationship of p19^(ARF) to p53-related processes ofapoptosis and transactivation. Two well-established systems were used togain insight into the normal function of p19^(ARF) and to determine itscapacity to influence p53-dependent activities. In the first study, theinventors exploited the fact that loss of Rb function is associated withunchecked proliferation and ectopic apoptosis in lens fiber cells(Morgenbesser S D., et al., Nature 371, 72-74 (1994)). Significantly,this apoptotic response was shown to be dependent upon p53, becauseembryos doubly null for Rb and p53 showed a 4- to 5-fold reduction inapoptotic events as measured by the TUNE assay (Morgenbesser S D., etal., Nature 371, 72-74 (1994)). The normal function of p19^(ARF) wasassessed by generating embryos doubly null for Rb and INK4a tospecifically evaluate p19^(ARF), as opposed to p16^(INK4a), function ina p53 apoptotic pathway. This line of reasoning based upon the findingthat p16^(INK4a), operates upstream of Rb and has no discernable growtheffects in Rb null cells (Serrano M., et al., Science 267, 249-252(1995); Medema R H., et al., Proc. Natl. Acad. Sci. USA 92, 6289-6293(1995); Lukas J., et al., Nature 375, 503-506 (1995)); and see FIG. 1Babove).

[0062] As shown in FIG. 4, histological analyses of many Rb−/− andRb−/−, INK4a−/− lenses staged 13.5 or 14.5 revealed a clear increase inthe number of nuclei compared with age matched wildtype lenses (comparepanels B or C with A). Moreover, doubly null lenses had a 25% increasein the number of nuclei are Rb−/− lenses (compare panels C and B). Whilethe lens fiber region of normal lenses does not exhibit proliferativeactivity ((Morgenbesser S D., et al., Nature 371, 72-74 (1994); panelD)), inappropriate cell cycle progression was confirmed throughout thelens fiber region of Rb−/−and Rb−/−, INK4a−/1-lenses by the large numberof cells positive for 5-bromo-2′-deoxyuridine (BrdU) (panels E and F).When normalized to the total number of nuclei, the degree of BrdUincorporation in Rb−/− and doubly null lens fiber cells was very similarin age-matched lenses (p<0.00l). In contrast, when lens fiber cellapoptosis was measured, the number of TUNEL-positive nuclei wassignificantly and consistently reduced in the doubly null lensesrelative to that present in the Rb-deficient lenses (compare panels Hand I; and see J for quantitation; p=0.003). The level of reductionachieved with loss of p19^(ARF) function was less than that reportedpreviously with loss of p53 ((Morgenbesser S D., et al., Nature 371,72-74 (1994)); reduction of 50 to 60% for Rb−/−, INK4a−/− versus 75 to85% for Rb−/−, p53−/−). These studies show that the efficient executionof an apoptotic response known to be dependent upon p53 requires INK4agene function, a function that is most likely served by p19^(ARF).

[0063] These findings, together with the cell transformation studies andMDM2 interaction, are consistent with the view that p19^(ARF) mayfunction normally to suppress neoplasia via enhancing p53's ability toeliminate inappropriately cycling cells. Since many biologicalactivities of p53 are highly dependent upon its capacity to function asa sequence-specific transcription factor, we tested whether p19^(ARF)positively regulates p53 transactivation potential. 293T cells weretransfected with a fixed amount of a p53 expression construct and CATreporter bearing multimerized p53 binding sites (PG13CAT) and anincreasing amount of a p19^(ARF) expression construct. A progressiveincrease in the level of CAT activity was observed upon the addition ofp19^(ARF) in a dose-dependent manner (FIG. 4C). Moreover, the additionof p19^(ARF) to Myc reporter assays did not affect Myc transactivationpotential (data not shown). The ability of p19^(ARF) to enhance p53transactivation potential and the importance of this property of p53 inp53-mediated tumor suppression provides a possible basis for theanti-oncogenic actions of p19^(ARF).

[0064] Distinct and cooperative effects of p16^(INK4a) and p19^(ARF) inthe suppression of primary cell transformation. The anti-oncogenicpotencies of the two ink4a gene products were tested in the rat embryofibroblast (REF) cotransformation assay (Land et al., 1983, Nature 304,596-602) against various oncogene combinations (e.g., Myc/RAS, E1a/RASor SV40 Large T Antigen (T-Ag)/RAS). This approach has been usedextensively to provide a quantitative measure of anti-oncogenic activityand allow for placement of these activities along known growth controlpathways (Lahoz et al., 1997, PNAS USA Jun. 7. 91, 5503-5507;Schreiber-Agus et al., 1995, Cell 80, 777-786; Alland et al., 1997,Nature 1997. May. 1. 387, 49-55). In the first series of experiments,the degree of inhibition of E1a/RAS-versus Myc/RAS-induced fociformation by p19^(ARF), p16^(Ink4a), or both was investigated. As shownin FIG. 6, addition of mouse p16^(Ink4a) induced a 1.7- to 3-foldreduction in foci number when added to c-myc/RAS transfections (panel A,p16^(INK4a)) and failed to cause a statistically significant decrease inE1a/RAS foci counts (panel B, p16^(INK4a)); these results are identicalto our previous report for the human p16^(Ink4a) (Serrano et al., 1995,Science 267, 249-252). Since E1A inactivates the Rb protein, the failureof p16^(INK4a) to suppress E1/RAS transformation is as expected (Serranoet al., 1995, Science 267, 249-252; Medema et al., 1995, Proc. Natl.Acad. Sci. USA 92, 6289-6293; Lukas et al., 1995, Nature 375, 503-506).In the same cotransfection experiments, addition of p19^(ARF) resultedin marked foci reductions in c-myc/RAS (5- to 10-fold) as well asE1a/RAS (4- to 5-fold) cotransfections (panels A and B, p19^(ARF)).E1a/RAS inhibition by p19^(ARF) was not further augmented by theaddition of p16^(Ink4a) (panel B, compare p19^(ARF) andp16^(INK4a)+p19^(ARF)). In contrast, co-addition of p16^(Ink4a) andp19^(ARF) resulted in a complete inhibition of c-myc/RAS transformation.Thus, the distinct activity profiles of p16^(INK4a) and p19^(ARF) (i.e.,E1a/Ras transfections), together with their additive effects in thec-myc/RAS transfections, suggest that these proteins suppress neoplasiathrough separable but cooperative mechanisms of action (see below).

[0065] Functional p53 is required for full oncogenic suppression byp19^(ARF). The cell cycle inhibitory effects of p19^(ARF) in primary MEFcultures have been shown to be p53-dependent (Kamijo et al., 1997a, Cell91, 649-659). To examine the possibility that p19^(ARF) may also act ina p53-dependent manner to suppress cellular transformation, theinventors employed cells rendered functionally (T-Ag ordominant-negative p53) or genetically (p53−/−) deficient for p53 intransformation assays. The addition of p19^(ARF) to T-Ag/RAScotransfections was found to have no effect on the number of focigenerated in the REF assay (FIG. 6C) or on the morphological/growthcharacteristics of these foci (data not shown). Since T-Ag is known toengage many pathways beyond p53 (Fanning, 1992, Journal of Virology 66,1289-1293; Van Dyke, 1994, Sem. Cancer Biol. 5, 47-60), the ability ofp19^(ARF) to suppress transformation in two other contexts was thenassessed. First, in comparison to the addition of an empty vectorcontrol, the addition of p19^(ARF) did not affect the number of focigenerated cotransfection of a dominant-negative mutant form of p53 (p53KH215) and RAS in the REF assay (FIG. 6C). Second, potentp19^(ARF)-induced suppression of Myc/RAS foci formation was observed inearly passage Ink4a−/− mouse embryonic fibroblasts (MEFs), but thissuppression was completely eliminated in MEFs doubly null for Ink4a andp53 (FIG. 6D). These results strongly suggest that p19^(ARF) does notact in a non-specific cytotoxic manner to reduce foci formation in theMyc/RAS and E1a/RAS experiments described above. Instead, these resultsappear to assign specificity to the anti-oncogenic actions of p19^(ARF).More specifically, in accord with the recently reported cell cyclestudies (Kamijo et al., 1997a, Cell 91, 649-659), these findings supportthe hypothesis that p19^(ARF) acts in a p53-dependent manner to inhibitcellular transformation.

[0066] p19^(ARF) associates with MDM2 in vivo. To gain insight into themechanistic basis for the functional link between p19^(ARF) and the p53pathway, co-immunoprecipitation experiments were performed to assesspotential physical interactions between p19^(ARF) and p53 or thep53-associated protein, MDM2. Since endogenous levels of these proteinsare very low in normal primary cells (Levine, 1997, Cell 88, 323-331),the composition of the p19^(ARF) complexes was determined followingco-transfection of various expression constructs (including one encodinga FLAG epitope-tagged p19^(ARF) protein, p19^(FLAG)) or through the useof different tumor cell lines expressing some or all of these proteins.As shown in FIG. 7A, IP-Western blot assays readily detected p19^(FLAG)in anti-p53 immunoprecipitates following co-transfection with p53, MDM2and p19FLAG (lane 2) but not with p53 and p19^(FLAG) (lanes 3 and 4).The requirement of MDM2 over-expression to reveal a p53-p19^(FLAG)interaction was also observed following either anti-p53 or anti-FLAGimmunoprecipitations of metabolically labeled transfected cells (datanot shown). These results demonstrate that p53, MDM2 and p19^(FLAG) canexist as components of a multi-protein complex in vivo. Moreover, therequirement for abundant MDM2 to detect p53-p19^(FLAG) interactionsuggested that MDM2 serves as a bridging molecule, or that MDM2 induceschanges in steady-state levels of p19^(FLAG), among other possibilities.The possibility that MDM2 overexpression stabilizes the level ofp19^(FLAG) was ruled out by Western blot analysis showing equivalentlevels of p19^(FLAG) in 293T cells following transfection of MDM2 andp19^(FLAG) or of p19^(FLAG) alone (data not shown). Moreover, althoughMDM2 can target p53 for degradation in some cell types (Haupt et al.,1997, Nature 387, 296-299; Kubbutat et al., 1997, Nature 387, 299-303),the levels of endogenous p53 in 293T cells remain constant followingco-transfection and over-expression of MDM2 (data not shown) due to thepresence of T-Ag (Henning, et al., 1997, Journal of Virology 71,7609-7618).

[0067] To examine more directly whether p19^(FLAG) can associate withMDM2, co-immunoprecipitation studies were conducted in metabolicallylabeled 293T cells and in 3T3DM (amplified for MDM2) and SAOS2 cells(null for p53). In the 293T cells (FIG. 7B and C), MDM2 was readilydetected in anti-FLAG immunoprecipitates following co-transfection withp19^(FLAG) and MDM2 (lane 8) and but not with either empty vector (lane6), p19^(FLAG) alone (lane 5) or MDM2 alone (lane 7). Correspondingly,anti-MDM2 immunoprecipitations confirmed the MDM2-p19^(FLAG) associationin the p19^(FLAG) and MDM2 co-transfections (lane 12). In addition, theendogenous p19^(ARF) band was present in the anti-MDM2immunoprecipitates (lane 9) and the signal intensity of this banddiminished upon co-transfection of p19^(FLAG) (lane 12), this likely dueto competition for a common binding site in the MDM2 complex. In each ofthese experiments, Western blot analyses of lysates that were run inparallel confirmed the identity of p19^(FLAG) and MDM2 bands (data notshown). The interaction between p19^(FLAG) and MDM2 in 293T cells wasalso demonstrated by co-immunoprecipitation in both low and highstringency conditions yielding identical results (FIG. 7C, lanes 18 and19, respectively).

[0068] To address whether the interaction of T-Ag with MDM2 and p53(Brown et al., 1993, Mol. Cell. Biol. 13, 6849-6857) alters thecomposition of MDM2/p53/p19^(ARF) complexes in T-Ag expressing 293Tcells, the interaction between endogenous p19^(ARF) and MDM2 wasexamined in 3T3DM cell lines. These cells do not express T-Ag but doexpress high levels of p19^(ARF) (Quelle et al., 1995b, Cell 83,993-1000) and MDM2, the latter due to gene amplification (Cahilly-Snyderet al., 1987, Som. Cell Mol. Gen. 13, 235-244; Fakharzadeh et al., 1997,EMBO J. 10, 1565-1569). Of note, 3T3DM cells express severalalternatively processed species of MDM2 protein (FIG. 7D, lane 20-21,p90/p85, p76/p74 and, in very low amounts, p57) (Barak, et al., 1994,Genes and Development 8, 1739-1749; Olson et al., 1993, Oncogene 8,2353-2360). Employing a p19^(ARF)-specific antibody forimmunoprecipitation, abundant MDM2 was readily detected upon Westernblotting of the immunoprecipitates with anti-MDM2 antibody (lane 21),further substantiating that p19^(ARF) and MDM2 interact in vivo.Moreover, the p76/p74 species which lacks the N-terminal p53 bindingdomain (Olson et al., 1993, Oncogene 8, 2353-2360) is also present inthe immunoprecipitates (lane 21, asterisk marks the p76/p74 forms ofMDM2 that do not interact with p53), suggesting that p19^(ARF) caninteract with MDM2 independent of MDM2's interaction with p53. Toconfirm this point, anti-FLAG immunoprecipitation followingco-transfection of p19^(FLAG) and MDM2 into p53 null SAOS2 cells yieldedabundant MDM2 signal (FIG. 7E, lane 26). Finally, confocal microscopicanalysis of the intracellular distribution of each protein demonstratedidentical subnuclear localization patterns for p19^(ARF) and MDM2 inboth 293T and 3T3DM cells (FIG. 7F, 293T cells shown).

[0069] Next, a series of plasmids encoding full-length MDM2 or variousmutant derivatives was tested for the ability to associate withFLAG-tagged p19^(ARF) by coimmunoprecipitation/Western blotting analysesin 293T and SAOS2 cells. The results of these studies, as shown in FIG.8, point to a complex interaction profile in which p19^(FLAG) engagesmultiple sites within MDM2. Specifically, the p19^(FLAG)-MDM2interaction was preserved with deletion of the entire carboxy terminalhalf of MDM2 (221-491, lanes 12-13) but this interaction was abolishedwith a slightly larger deletion (155-491, compare lysate in lane 10 toimmunoprecipitation in lane 11). While these observations demonstrate anessential role for MDM2 amino acid residues 154-221 in p19^(ARF)binding, an internal deletion mutant of these residues was still capableof p19^(ARF) association in the two cell types (155-221, lanes 6-7 293T,lanes 8-9 SAOS2). The persistent binding of the 155-221 mutant mayreflect additional points of contact in the carboxy terminus of MDM2that cooperate in p19^(ARF) binding. This view is supported by thediminished interaction between p19^(FLAG) and the 221-491 mutant inSAOS2 cells (compare lanes 12, 13 to 14, 15) and also suggests theparticipation of bridging molecules in 293T that facilitate thep19^(FLAG)-MDM2 interaction (e g., p53,T-Ag, etc.). Taken together, thecarboxy-terminal localization, coupled with the ability of a p53-bindingdeficient mutant of MDM2 to remain competent for p19^(ARF) binding (lane1), suggest that p19^(ARF)'s effects upon known properties of MDM2 orp53 (see below) likely do not result from a disruption of the physicalassociation between MDM2/p53 by p19^(ARF). Further support thatp19^(ARF) and p53 interact with non-overlapping regions of MDM2 comesfrom the observations that p19^(ARF) and p53 can co-exist in MDM2complexes and that p53 immunoprecipitations followed by Western analysisfor MDM2 showed similar levels of MDM2 relative to lysate in thepresence or absence of p19^(ARF) (data not shown).

[0070] Functional relationship of p19^(ARF) to MDM2 and p53. Thephysical association between p19^(ARF) and MDM2 establishes a clearconnection between a product of the INK4a gene and the p53 pathway. Tounderstand the functional implications of the p19^(ARF)-MDM2interaction, the capacity of p19^(ARF) to (1) inhibit MDM2cotransformation activity, (2) block MDM2-induced degradation of p53,and (3) enhance p53-related activities such as transcription andapoptosis was assessed.

[0071] Transformation Studies. For the MDM2 transformation studies, theinventors took advantage of the capacity of MDM2 to cooperate withactivated RAS to effect the malignant transformation of early passageREFs (Finlay, 1993, Molecular & Cellular Biology 13, 301-306). In fourindependent experiments, the inventors observed that the addition ofp19^(ARF) to MDM2/RAS cotransfections resulted in a dramatic reductionin foci numbers, e.g., 40 foci versus 3 foci (FIG. 9A). Moreover,compared with MDM2/RAS and vector controls, the MDM2/RAS transformedfoci emerging in the p19^(ARF) cotransfections exhibited a lesstransformed morphology (data not shown).

[0072] p53 protein stability studies. Next, we examined the consequencesof p19^(ARF) over-expression on a key biochemical property of MDM2,namely MDM2's ability to promote the rapid degradation of p53 (Haupt etal., 1997, Nature 387, 296-299; Kubbutat et al., 1997, Nature 387,299-303). For this study, HeLa cells were transiently transfected withthe various expression constructs listed in FIG. 9B and the levels ofp53 were examined by Western blot analysis. As reported previously(Haupt et al., 1997, Nature 387, 296-299), p53 steady-state levels weremarkedly reduced in cells co-transfected with p53 and MDM2 as opposed top53 alone (FIG. 9B, top panel, compare lanes 2 and 3). When p19^(ARF)was added to the p53+MDM2 co-transfections, a striking restoration inp53 levels was observed (lane 4). Equal loading of protein was confirmedby reprobing the blots with an anti-FLAG antibody, which detects anon-specific background band (NSFE) (FIG. 9B, bottom panel) as well asby Ponceau Red staining of blots (data not shown). The precise mechanismthrough which p19^(ARF) operates to interfere with MDM2-induceddegradation is not known. Nevertheless, it is interesting that MDM2induces a ladder of more slowly migrating bands of p53 (FIG. 9C, lane1), thought to represent ubiquitinated forms of p53 bound forproteasomal degradation (Haupt et al., 1997, Nature 387, 296-299;Kubbutat et al., 1997, Nature 387, 299-303). This ladder issignificantly reduced in the presence of abundant p19^(ARF) (FIG. 9C,lane 2), suggesting that p19^(ARF) inhibits polyubiquitination of p53triggered by MDM2. Loss of this MDM2-induced ladder was also observedafter transfection of p19^(ARF), p53 and MDM2 into two other cell lines,H1299 and SAOS2 (data not shown). Although our studies strongly suggestthat p19^(ARF) blocks MDM2-induced degradation of p53, they do notexclude other possibilities such as p19^(ARF) stabilizing p53 in anMDM2-independent manner.

[0073] Regulation of p53 transactivation activity. Enforced expressionof p19^(ARF) in primary mouse cells results in the induction ofp21^(CIP1) (a p53-responsive gene (El-Deiry et al., 1993, Cell 75,817-825)), but only if these cells that possess functional p53 (Kamijoet al., 1997a, Cell 91, 649-659) (and JP and RD, unpublishedobservations) These results suggest that p19^(ARF) can enhance thetransactivation activity of p53 (Kamijo et al., 1997a, Cell 91,649-659), perhaps through its ability to counteract MDM2. To test thisdirectly, SAOS2 cells were transfected with a CAT reporter bearingmultimerized p53 binding sites in its promoter and with a combination ofexpression constructs listed in FIG. 9D. Since these cells are null forp53, CAT activity was detected only in the presence of transfected p53(compare lane 1 with lanes 2 and 3). Transfection of p19^(ARF) resultedin a further increase in reporter gene activity (lanes 4 and 5), anincrease that takes place in the presence of detectable endogenous MDM2levels resulting from exogenous p53 expression (data not shown). Asreported previously (Brown et al., 1993, Mol. Cell. Biol. 13, 6849-6857;Momand et al., 1992, Cell 69, 1237-1245), addition of MDM2 to the p53cotransfections led to a decrease in reporter activity (compare lanes 6and 7 with lanes 2 and 3) and this effect was abolished with theaddition of p19^(ARF) (lanes 8 and 9).

[0074] In light of the data presented above, enhanced p53transactivation could result from increased p53 levels due top19^(ARF)-induced stabilization of p53. However, the findings thatover-expression of p19^(ARF) leads to stabilized p53 complexes whichalso contain MDM2 (FIG. 7) and that MDM2 binds to and masks the p53transactivation domain raises questions as to how p53 transactivationcan be restored by p19^(ARF) in the setting of high levels of MDM2.Among several possibilities are that a subset of transactivation domainsin the stabilized p53 tetramer are not bound by MDM2, that p19^(ARF) mayfunction to block MDM2-induced repression of basal transcription, orthat p19^(ARF) activates p53 transactivation in an MDM2—independentmanner. The resolution of this point will require further analysis invitro. In summary, these results demonstrate that p19^(ARF) can enhancea key function of p53, its capacity to function as a sequence-specifictranscription factor.

[0075] INK4a-deficiency attenuates apoptosis in vivo. The observedeffects of p19^(ARF) on p53 dependent transactivation (this study) andgene expression (Kamijo et al., 1997, Cell 91, 649-659), and theestablished importance of p53 in apoptosis prompted us to assess whetherloss of p19^(ARF) may affect the degree of apoptosis in vivo. Theinventors have shown previously that the developing mouse lensrepresents an ideal system for such an analysis since loss of Rbfunction therein is associated with unchecked proliferation andapoptosis in lens fiber cells and this apoptotic response is highlydependent upon p53 (Morgenbesser et al., 1994, Nature 371, 72-74). As anindirect assessment of p19^(ARF) effects upon this phenotype, rates ofproliferation and apoptosis were compared in embryos singly null for Rbor doubly null for Rb and INK4a. Since p16^(INK4a) is believed to bewithout effect when Rb is absent, the doubly null lenses were taken tobe the functional equivalent of Rb−/−, p19^(ARF)−/− lenses.

[0076] Histological analyses of more than 15 Rb−/− and Rb−/−, ink4a−/−lenses revealed a clear increase in the number of nuclei compared withage-matched wildtype lenses (FIG. 10A, compare panels b and c with a).Moreover, doubly null lenses had a 25% increase in the number of nucleiover Rb−/− only lenses. While the lens fiber region of normal orink4a−/− lenses does not exhibit proliferative activity (Morgenbesser etal., 1994, Nature 371, 72-74) (FIG. 10A, panel d, ink4a−/− not shown),inappropriate cell cycle progression was confirmed throughout the lensfiber region of Rb−/− and Rb−/−, ink4a−/− lenses by the large number ofcells staining positive for 5-bromo-2′-deoxyuridine (BrdU) incorporation(FIG. 10A, panels e and f) When normalized to the total number ofnuclei, the degree of BrdU incorporation in Rb−/− and doubly null lensfiber cells was very similar in age-matched lenses (FIG. 10B left panelp<0.001). In contrast, when lens fiber cell apoptosis was measured, thenumber of TUNEL-positive nuclei was significantly and consistentlyreduced in the doubly null lenses relative to that present in theRb-deficient lenses (FIG. 10A, compare panels h and i; FIG. 10B rightpanel). These studies show that the efficient execution of an apoptoticresponse known to be dependent upon p53 requires full ink4a genefunction. The dual elimination of both ink4a gene products precludes adefinitive assignment to p19^(ARF) since it remains theoreticallypossible that p16^(INK4a) may play a role in the apoptotic processthrough an Rb-independent pathway. However, these findings may explainhow p19^(ARF) functions as a suppressor of neoplasia, namely through itscapacity to enhance the p53-mediated elimination of inappropriatelycycling cells in vivo.

[0077] III. Discussion

[0078] Analysis of tumor associated mutations affecting the INK4a locusin mouse (data not shown) and humans (Kamb A., et al., Science 264,436-440 (1994)) has revealed a high incidence of homozygous co-deletionof p16^(Ink4a) and p19^(ARF) sequences. These observations, togetherwith the functional and physical evidence presented here, lead us topropose that p19^(ARF) contributes to the anti-oncogenic activity ofINK4a and that the frequent elimination of both INK4a gene productsreflects a requirement to disable two functionally distinct growthinhibitory pathways. From a mechanistic standpoint, full oncogenicsuppression by p19^(ARF) requires p53 as evidence by a significantreduction in p19^(ARF) activity in the presence of SV40 TAg or adominant negative mutant of p53 or in the absence of p53 (p53−/− MEFs).The ability of p19^(ARF) to enhance p53-related functions in theRb-deficient lens (apoptosis) and in reporter assays (transactivation)suggests that p19^(ARF) acts as an activator of p53 activity. Based uponthe existence of a p19^(ARF)-MDM2 interaction in cells, it is temptingto speculate that p19^(ARF) could function to neutralize MDM2-inducedinhibition of p53. However, it is important to point out that p19^(ARF)also associates with the form of MDM2 (p76/p74) which lacks theN-terminal p53 interaction pocket, suggesting mechanisms other than acompetitive occupation of the p53 interaction pocket by p19^(ARF).Moreover, since other MDM2 species that interact with p19^(ARF) can alsointeract with p53, there may be additional interactions betweenp19^(ARF) and p53 in the absence of MDM2. While a detailed accounting ofthese interactions should provide important mechanistic clues, thefindings of this report are the first to establish a clear connectionbetween the ink4a gene and the p53 tumor suppressor pathway.

[0079] One-gene-two-products-two-pathways: Implications fortumorigenesis. The potential to disrupt two essential growth controlpathways through a single genetic hit may provide an explanation for:(i) the exceedingly high rate of Ink4a gene deletion in may human tumorsand their derivative cell lines (Kamb A., et al., Science 264, 436-440(1994)); (ii) the high incidence of spontaneous tumors in mice lackingInk4a exon 2/3 sequences (Serrano, M., et al., Cell 85, 27-37 (1996));and (iii) the strong connection between tumorigenesis and the Ink4a geneas opposed to other genes encoding cyclin-dependent kinase inhibitors,such as INK4b, p21^(CIP1) and p27^(KIP120) Specifically, mice lackingINK4b exhibit a very low incidence of spontaneous tumor formation (E.Latres, C. Cordon-Cardo and M. Barbacid, unpublished),p21^(CIP1)-deficient mice remain tumor free (Elledge S J., et al., TICB6, 388-392 (1996)), and although p27^(KIP2)-deficient mice can developintermediate lobe pituitary hyperplasia or adenoma, these neoplasmararely progress to malignant pituitary tumors (Elledge S J., et al.,TICB 6, 388-392 (1996)). Similarly, in human cancers, the frequentalteration of ink4a contrasts sharply with an overall lower rate ofink4b mutation/deletion (Cordon-Cardo C., Am. J. Pathol. 147, 545-560(1995)) and infrequent mutations in p21^(CIP1) and p27^(KIP120) Suchbiological correlates would not have been anticipated in view of thehighly similar biochemical and growth suppressive activities of thesecyclin-dependent kinase inhibitors. What makes the INK4a gene so unique?Based upon the findings of this study, we propose that INK4a's potenttumor suppressor activity results from its ability to encode twodifferent anti-oncogenic proteins with cooperating modes of action. Oneprediction of this hypothesis is that tumors deficient for bothp16^(Ink4a) and p19^(ARF) would be less likely to harbor Rb or p53mutations. Furthermore, p19^(ARF)-sparing ink4a mutations could beassociated with alterations involving other components of the p53pathway (e.g., MDM2 amplification or loss of p53 function). It isimportant to emphasize that elimination of p19^(ARF) would not precludep53 mutation since p53 plays multiple roles in suppressing neoplasticgrowth that are likely to extend beyond the p19^(ARF)-p53 connection.Stated differently, loss of function mutations of p19^(ARF) would bepredicted to decrease the frequency of, but not eliminate,tumor-associated p53 mutations.

[0080] In light of the above observation, the inventors re-examinedreported Ink4a and p53 mutations in the same human cancers (Gruis N A.,et al., Am. J. Pathol. 146, 1199-1206 (1995); Hangaishi A., et al.,Blood 87, 4949-4958 (1996); Heinzel P A., et al., Intl. J. Cancer 68,420-423 (1996); Kinoshita I., et al., Cancer Res. 56, 5557-5562 (1996);Newcomb E W., et al., Mol. Carcin 14, 141-146 (1995); Brenner A., etal., Clin. Cancer Res. 2, 1993-1998 (1996)). These tumor types includedmelanoma, carcinomas (bladder, oral, and lung carcinomas), and variouslymphoid neoplasms (B-cell chronic lymphocytic leukemia, Hodgkin andNon-Hodgkin lymphomas). Our analysis demonstrated a reciprocalrelationship between these two genes, since Ink4a-deficient(p16^(Ink4a)+p19^(ARF)) cancers rarely exhibit p53 mutant products(Gruis N A., et al., Am. J. Pathol. 146, 1199-1206 (1995); Hangaishi A.,et al., Blood 87, 4949-4958 (1996); Heinzel P A., et al., Intl. J.Cancer 68, 420-423 (1996); Kinoshita I., et al., Cancer Res. 56,5557-5562 (1996); Newcomb E W., et al., Mol. Carcin 14, 141-146 (1995);Brenner A., et al., Clin. Cancer Res. 2, 1993-1998 (1996)). In 518tumors analyzed, the mutation rates were 18% for p16^(INK4a), 14% forp53, and 4% for both. Ink4a point mutations that result in single aminoacid changes in the p16^(INK4a) ORF were reanalyzed to determine thegenetic status of the p19^(ARF) ORF. Only 9 (<2%) of 405 evaluable casesharbored p53 and p19^(ARF) mutations. Since all 9 cases also hadalterations in p16^(INK4a), it remains possible that p19^(ARF) mutationswas incidental to that of p16^(INK4a) in those tumors. Moreover, theimpact of these p19^(ARF) mutations on p19^(ARF) function remains to bedetermined. These observations point to the need for a revisitedanalysis of mutations for p19^(ARF), p16^(INK4a), p53 and MDM2 in thesame tumor samples. In fact, the most common point mutation in thep19^(ARF) reading frame (a P93L substitution (Quelle et al., 1995b,Oncogene 11, 635-645)) has been shown by the inventors to befunctionally indistinguishable from wild-type p19^(ARF). This isevidenced by the facts that the p19^(ARF) (P93L) mutant is fully activein suppressing Myc/RAS and MDM2/RAS in the REF assay and in stabilizingp53 in the presence of high MDM2 levels (data not shown). Theseobservations point to the need for a revisited analysis of mutations forp19^(ARF), p16^(INK4a), p53 and MDM2 in the same tumor samples. Althoughavailable data (Kamijo et al., 1997a, Cell 91, 649-659) (and this paper)do not permit conclusion that p19^(ARF) and p53 mutations are mutuallyexclusive, the distinctly uncommon occurrence of co-mutation of p53 andp19^(ARF) supports the view that they operate through common geneticpathways for at least a significant portion of their tumor suppressoractivity.

[0081] Therapeutic implications. The studies by the inventorsdemonstrating that dominant interference with p53 nullifies p19^(ARF)suppression presage that the introduction of p19^(ARF) into tumorsrendered functionally or genetically deficient for p53 would fail torespond clinically. Such an outcome agrees well with the observations ofQuelle et al. (Quelle D E., et al., Cell 83, 993-1000 (1995)), whoreported high levels of p19^(ARF) mRNA only in p53-null cell lines,suggesting a functional interrelationship between these two proteins. Byextension, an improved therapeutic outcome in p53-null tumors would beexpected with regimens that included both p53 and p19^(ARF) as opposedto p53 alone. Finally, in tumors null for ink4a or for both Rb and p53,the addition of p16^(Ink4a) to a p19^(ARF)/p53 combination may actuallydiminish the efficacy of p19^(ARF)/p53 based upon the potent growtharresting activity of p16^(INK4a) and a possible diminution inelimination of tumor cells by programmed cell death. In agreement withthis prediction are recent studies demonstrating thatp16^(Ink4a)-mediated cell cycle arrest resulted in a dramatic increasein resistance to chemotherapeutic agents (Stone S., et al., Cancer Res.56, 3199-3202 (1996)).

[0082] The connection to the RB and p53 pathways has importantimplications for understanding the prominent role served by INK4a incellular growth, survival, senescence and neoplasia. In normal cells,the capacity of a single gene to encode regulations of both RB and p53may enable the coordination of two pathways known to be integral to theentry of normal cells into replicative senescence. In cancer cells, theloss of two distinct tumor suppressor products with the elimination of asingle gene would provide a strategic genetic route to neoplasia and, assuch, may account for the frequent-involvement of INK4a in thedevelopment of a broad spectrum of malignancies.

[0083] Recent studies in cell culture and with knockout mouse modelshave determined that the second product of the Ink4a locus, namelyp19^(ARF), functions as a potent growth and tumor suppressor that exertsits actions upstream of p53 (Kamijo et al., 1997a, Cell 91, 649-659;Quelle et al., 1997, Proc. Natl. Acad. Sci. USA 94, 669-673; Chin etal., 1997, Genes and Development 11, 2822-2834). Here the inventorsrefined this connection between p19^(ARF) and the p53 pathway bydemonstrating that p19^(ARF) physically associates with MDM2 in vivo andblocks MDM2-induced degradation of p53. The end result of these actionsappears to be the enhancement of p53-related functions such astransactivation (reporter assays, FIG. 9D), growth inhibition (Kamijo etal., 1997a, Cell 91, 649-659) and possibly apoptosis (lens studies, FIG.10) (see model in FIG. 11B). The inventors believe that their studiesprovide genetic evidence, in addition to physical data, that p19^(ARF)acts primarily on the level of MDM2 rather than p53. The conceptualbasis for this argument rests on the fact that although MDM2over-expression acts to neutralize p53, p19^(ARF) can still inhibitoncogenesis in this setting (FIG. 9A). In contrast, other oncoproteinsthat can neutralize p53 render cells refractory to p19^(ARF) suppression(FIG. 6B). Thus, the inventors propose that either (i) p19^(ARF) caninterfere with the ability of MDM2 to neutralize p53 (FIG. 11B) or (ii)p19^(ARF) can affect as yet undetermined MDM2-specific transformationfunctions beyond those regulating p53 levels and activity. The latter,although formally possible, appears less likely in light of mouseknockout studies suggesting that MDM2 functions primarily (if notexclusively) as a modulator of p53 function (Jones et al., 1995, Nature378, 206-208). Regardless of the precise mechanism, the physical andfunctional link forged between p19^(ARF) and the p53 pathway, along withthe previously established one between p16^(INK4a) and Rb (Quelle etal., 1995a, Oncogene 11, 635-645; Serrano et al., 1993, Nature 366,704-707) provide for a “one-gene-two products-two pathways” hypothesis(FIG. 11A) that can explain (i) the exceedingly high rate of INK4a genedeletion in many human tumors and their derivative cell lines (Kamb etal., 1994, Science 264, 436-440) and in mouse melanomas (Chin et al.,1997, Genes and Development 11, 2822-2834) and (ii) the strongconnection between tumorigenesis and the INK4a gene as opposed to othergenes encoding cyclin-dependent kinase inhibitors (CKIs), such as INK4b,p21^(CIP1) and p27^(KIP1) (Cordon-Cardo, 1995, Am. J. Pathol. 147,545-560). Specifically, mice lacking INK4b exhibit a very low incidenceof spontaneous tumor formation (E. Latres, C. Cordon-Cardo, and M.Barbacid, unpublished), p21^(CIP1)-deficient mice remain tumor-free(Elledge et al., 1996, TICB 6, 388-392), and althoughp27^(KIP1)-deficient mice can develop intermediate lobe pituitaryhyperplasia or adenoma, these neoplasms rarely progress to malignantpituitary tumors (Elledge et al., 1996, TICB 6, 388-392). Similarly, inhuman cancers, the frequent alteration of INK4a contrasts sharply withan overall lower rate of INK4b mutation/deletion (Cordon-Cardo, 1995,Am. J. Pathol. 147, 545-560) and infrequent mutations in p21^(CIP1) andp27^(KIP1) (Cordon-Cardo, 1995, Am. J. Pathol. 147, 545-560). What makesthe INK4a gene so unique among the CKIs with respect to tumorigenesis?In essence, two functionally distinct tumor suppressor pathways can bedisabled by a single mutational event at the INK4a locus, this by virtueof its unique genetic organization. Stated differently, INK4a's potenttumor suppressor activity likely results from its ability to encode twounrelated anti-oncogenic proteins with cooperating modes of action (FIG.11A).

[0084] Based upon the documented growth arresting activity encoded bythe INK4a gene products (Serrano et al., 1993, Nature 366, 704-707;Quelle et al., 1995b, Cell 83, 993-1000) and the reduction in apoptosisin the lenses doubly null for Rb and INK4a (see FIG. 10 above), theinventors suggest that the mechanisms of tumor suppression by INK4aparallel those established for Rb and p53. Specifically, thecollaborative consequences of loss of p16^(INK4a) and p19^(ARF) arederegulated cell proliferation as well as deactivation or attenuation ofp53-dependent apoptosis which normally serves to promote the efficientelimination of these pre-malignant cycling cells. In agreement with thishypothesis is the observation that tumors arising in INK4a-deficientmice exhibit high proliferative indices and very low rates of apoptosisdespite an intact p53 gene (CCC and RD, unpublished observations).

[0085] One prediction of this hypothesis is that tumors deficient forboth p16^(Ink4a) and p19^(ARF) would be less likely to harbor Rb or p53mutations. Furthermore, p19^(ARF)-sparing INK4a mutations could beassociated with alterations involving other components of the p53pathway (e.g., MDM2 gene amplification or loss of p53 function). It isimportant to emphasize that elimination of p19^(ARF) may not precludep53 mutation since p19^(ARF) tumor suppressor activities are unlikely tooverlap fully with those of p53. This lack of equivalence is madeevident by the much higher level of genetic instability in p53−/− MEFscompared with INK4a−/− MEFs (Kamijo et al., 1997) (and NL and RD,unpublished) and by the higher rate of spontaneous tumor formation inp53−/− mice versus Ink4a−/− mice (Serrano et al., 1996, Cell 85, 27-37;Jacks, 1994). In the lens, the level of reduction in apoptosis achievedwith loss of Ink4a function was less than that reported previously withloss of p53 (Morgenbesser et al., 1994) (reduction of 50 to 60% forRb−/−, Ink4a−/− versus 75 to 85% for Rb−/−, p53−/−) Notwithstanding,loss of function mutations of p19^(ARF) would be predicted to decreasethe frequency of tumor-associated p53 mutations.

1 2 1 29 DNA Artificial Sequence primer for MDM2 mutant 1 cgccatctagaccggatctt gatgctggt 29 2 18 DNA Artificial Sequence primer for MDM2mutant 2 cgaagggccc aacatctg 18

What is claimed:
 1. A method for inhibiting growth of a tumor cellcomprising introducing to the cell an effective amount of p19^(ARF) or amimetic thereof, and p53 to inhibit growth of the tumor cell.
 2. Themethod of claim 1, wherein the p19^(ARF) or a mimetic thereof, and p53are introduced to the cell by administering p19^(ARF) or a mimeticthereof, and p53 proteins to said cell.
 3. The method of claim 1,wherein the p19^(ARF) or a mimetic thereof, and p53 are introduced tothe cell by administering p19^(ARF) proteins, or a mimetic thereof, andnucleic acid encoding p53 to said cell.
 4. The method of claim 1,wherein the p19^(ARF) or a mimetic thereof, and p53 are introduced tothe cell by introducing nucleic acid encoding p19^(ARF) or a mimeticthereof, and p53 to the cell, so that the nucleic acid encodingp19^(ARF) or a mimetic thereof, and p53 is expressed in amountseffective to inhibit growth of the tumor cell.
 5. The method of claim 1,wherein the p19^(ARF) or a mimetic thereof, and p53 are introduced tothe cell by introducing nucleic acid encoding p19^(ARF) or a mimeticthereof, and p53 protein to the cell, so that the nucleic acid encodingp19^(ARF) or a mimetic thereof is expressed in amounts effective toinhibit growth of the tumor cell.
 6. A pharmaceutical compositioncomprising p19^(ARF) or a mimetic thereof.
 7. The pharmaceuticalcomposition of claim 6, further comprising p53.
 8. The pharmaceuticalcomposition of claim 7, further comprising p16^(Ink4a).
 9. Apharmaceutical composition comprising nucleic acid encoding p19^(ARF) ora mimetic thereof.
 10. The pharmaceutical composition of claim 9,further comprising nucleic acid encoding p53.
 11. The pharmaceuticalcomposition of claim 10, further comprising nucleic acid encodingp16^(Ink4a).